Composition including rilpivirine and method for treating tumors or cancer

ABSTRACT

The present invention provides a composition and method for preventing the growth of and/or treating cancerous tumors and/or delaying onset of cancer from tumor-initiating cells. The composition includes an effective amount of a compound of Formula (I) or Formula (II):or any pharmaceutically acceptable salt thereof. The composition is administered alone or in combination with one or more chemotherapeutic agents, biological agents and/or anticancer agents. The method may include administering the composition of the present invention with or without the chemotherapeutic, biological, or other cancer treatment agent to a subject in need thereof intravenously, parenterally, nasally, topically or locally, orally, or by liposome, implant or via vessel-targeted nanosuspension delivery.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit under 35 U.S.C. § 119(e) ofU.S. Provisional Application Nos. 62/941,891, filed Nov. 29, 2019, and63/072,281, filed Aug. 31, 2020, the disclosures of each of which areincorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to a composition comprising rilpivirinefor cancer treatment as well as treatment of precancerous conditions. Inparticular, the composition is administered alone or in combination withone or more therapeutic agents comprising one or more ofchemotherapeutic, biological and/or anticancer agents to a subject inneed thereof.

BACKGROUND

Cancer is a complex disease which is usually caused by specific changesto the genes in one cell or group of cells and leads to disruption innormal cell function. There are at least six major differences betweennormal cells and cancer cells. (A) Growth: normal cells stop growingwhen sufficient cells are present. However, cancer cells do not stopgrowing and divide more quickly than normal cells; this uncontrolledcell growth may result in the formation of a tumor. (B) Cell repair andcell death: normal cells are either repaired or experience programmedcell death when they are damaged or older (apoptosis). In contrast,cancer cells are either not repaired or do not undergo apoptosis. (C)Metastasis: normal cells stay in the region of the body where theybelong; however, cancer cells migrate and invade other tissues or organsvia the bloodstream and the lymphatic system. (D) Appearance: normalcells have a regular, ordered appearance; in contrast cancer cells aremisshapen, and appear as a chaotic collection of cells, in an array ofshapes and sizes. (E) Maturation: normal cells usually differentiateinto mature cells and functionalize naturally; however cancer cells growand divide rapidly and stay in an undifferentiated state. (F) Genomicstability: normal cells have standard DNA and chromosome numbers;however, the chromosomes of cancer cells often have abnormal numbers ofchromosome and cancer cells' DNA may become increasingly abnormal as itaccumulates mutations.

There are various approaches to cancer treatment. For many types ofcancer, chemotherapy is one of the most common and conventionaltreatments. In general, it involves administration of cytotoxic agentsto destroy cancer cells or stop them from growing/dividing and migratingto other parts of the body. More than 100 chemotherapy agents are usedfor cancer treatment. There are several different classes of drugsemployed during cancer treatment including: (a) alkylating agents whichprevent the cells from reproducing by damaging their DNA in all phasesof cell cycles; (b) anti-metabolites which interfere with thereplication of DNA and RNA by substituting for the normal buildingblocks of DNA and RNA; (c) antibiotics which inhibit the enzymes for DNAreplication; (d) topoisomerase inhibitors which inhibit enzymes forunwinding strands of DNA during replication and transcription, i.e.,topoisomerase I or topoisomerase II; (e) mitotic inhibitors whichinhibit mitosis and cell division; and (f) corticosteroids which areused for relieve the side effect caused by other drugs.

Chemotherapeutic agents inhibit the growth of cells including bothcancer cells and normal cells; these normal cells include new bloodcells in the bone marrow, epithelial cells in the mouth, stomach, skin,hair, or reproductive organs in the body. This is the major limitationor disadvantage of chemotherapy: it is unable to differentiate betweennormal and abnormal cells in the body. Therefore, patients oftenexperience serious side effects during and after chemotherapy treatment.

The anti-proliferative effects of non-nucleoside reverse transcriptaseinhibitors (NNRTIs) have been the subject of various studies. Forexample, certain reverse transcriptase inhibitors such as nevirapine andefavirenz have been shown to antagonize tumorigenic growth of A-375melanoma and PC3 prostate cancer cell lines in animal experiments(Oncogene 24:3923-3931, 2005). Dapivirine has been shown to inhibittumor growth of U87 glioblastoma cells (Journal of Cancer 9(1):117-128,2018). In vitro efavirenz and rilpivirine (a second generation NNRTIapproved by FDA for the treatment of HIV infection) have been found tohave high toxic potential against pancreatic cancer cells (PloS ONE10(6):e0130277, 2015). However, there remains a need for developing newcancer treatments, particularly those that are well-tolerated and havefewer serious side effects. More specifically, there remains a need foradditional medicines that function safely, effectively, and/orsynergistically in combination with other anticancer treatments, as wellas a need for cancer treatments that may be safe and effective withoutadditional chemotherapeutic agents.

SUMMARY OF THE INVENTION

The present invention is not to be limited in scope by any of thefollowing descriptions. The following examples or embodiments arepresented for exemplification only.

The term “cancer” not only refers to solid tumors, such as cancers ofthe breast, respiratory tract, brain, nerve tissue, reproductive organs,digestive tract, urinary tract, eye, liver, skin, head and neck,thyroid, parathyroid, and their distant metastases, but also bloodcancers including but not limited to lymphomas, sarcomas, and leukemias.

Breast cancers include, but are not limited to invasive ductalcarcinoma, invasive lobular carcinoma, ductal carcinoma in situ, andlobular carcinoma in situ.

Respiratory tract cancers include, but are not limited to small-cell andnon-small-cell lung carcinoma, as well as bronchial adenoma andpleuropulmonary blastoma.

Brain cancers include, but are not limited to brain stem andhypothalamic glioma, cerebellar and cerebral astrocytoma, glioblastoma,medulloblastoma, ependymoma, as well as neuroectodermal and pinealtumor.

Eye cancers include, but are not limited to intraocular melanoma andretinoblastoma.

Liver cancers include, but are not limited to hepatocellular carcinoma(liver cell carcinomas with or without fibrolamellar variant),cholangiocarcinoma (intrahepatic bile duct carcinoma), and mixedhepatocellular cholangiocarcinoma.

Skin cancers include, but are not limited to squamous cell carcinoma,Kaposi's sarcoma, malignant melanoma, Merkel cell skin cancer, andnon-melanoma skin cancer.

Head-and-neck cancers include, but are not limited tolaryngeal/hypopharyngeal/nasopharyngeal/oropharyngeal cancer, and lipand oral cavity cancer.

Lymphomas include, but are not limited to AIDS-related lymphoma,non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, Hodgkin's disease,and lymphoma of the central nervous system.

Sarcomas include, but are not limited to sarcoma of the soft tissue,fibrosarcoma, osteosarcoma, malignant fibrous histiocytoma,lymphosarcoma, and rhabdomyosarcoma.

Leukemias include, but are not limited to acute myeloid leukemia, acutelymphoblastic leukemia, chronic lymphocytic leukemia, chronicmyelogenous leukemia, and hairy cell leukemia.

Nerve tissue related tumor include, but are not limited toneuroblastoma, ganglioneuroblastoma, ganglioneuroma, schwannomas, orneurofibrosarcomas.

Male reproductive organ tumors include, but are not limited to prostateand testicular cancer.

Female reproductive organ tumors include, but are not limited toendometrial, cervical, ovarian, vaginal, and vulvar cancer, as well assarcoma of the uterus.

Digestive tract tumors include, but are not limited to anal, colon,colorectal, esophageal, gallbladder, gastric, pancreatic, rectal, smallintestine, and salivary gland cancers.

Urinary tract tumors include, but are not limited to bladder, penile,kidney, renal pelvis, ureter, and urethral cancers.

“Precancerous conditions” as used herein, relate to abnormal cells thathave an increased risk of turning cancerous.

Above diseases have been well characterized in humans, and can betreated by administering one or more therapeutic agents of the presentinvention.

A pharmaceutically acceptable excipient is any excipient which isrelatively non-toxic and innocuous to a patient at concentrationsconsistent with effective activity of the active ingredient so that anyside effects ascribable to the excipient do not vitiate the beneficialeffects of the active ingredient.

One aspect of the present invention provides a composition forinhibiting growth of and/or treating a cancerous tumor and/or delayingonset of cancer from tumor-initiating cells, the composition comprising:

an effective amount of a compound of Formula (I) or Formula (II):

or any pharmaceutically acceptable salt thereof, said composition beingadministered alone or in combination with one or more of the followingchemotherapeutic agents, biological agents and/or anticancer agents(“therapeutic agents” is used interchangeably hereinafter) to a subjectin need thereof.

In one aspect, the one or more chemotherapeutic agents may be alkylatingagents, anti-metabolites, antitumor antibiotics, topoisomeraseinhibitors, kinase inhibitors, mitotic inhibitors, steroids and/or anymixtures thereof.

The one or more biological agents may include but are not limited tovaccines, cytokines, antibodies, protein and peptide drugs, and/or anymixtures thereof. The treatment with rilpivirine may also be combinedwith other cancer treatments such as radiation treatment, T-celltherapy, etc.

In one embodiment, the composition of the present invention can becombined with anti-cancer agents including, but not limited toasparaginase, bleomycin, carboplatin, carmustine, chlorambucil,cisplatin, colaspase, cyclophosphamide, cytarabine, dacarbazine,dactinomycin, daunorubicin, doxorubicin (adriamycine), epirubicin,etoposide, 5-fluorouracil, hexamethylmelamine, hydroxyurea, ifosfamide,irinotecan, leucovorin, lomustine, mechlorethamine, 6-mercaptopurine,mesna, methotrexate, mitomycin C, mitoxantrone, prednisolone,prednisone, procarbazine, raloxifen, streptozocin, tamoxifen,thioguanine, topotecan, vinblastine, vincristine, and vindesine.

In one embodiment, the composition of the present invention can becombined with other cytotoxic drugs including, but not limited toaminoglutethimide, L-asparaginase, azathioprine, 5-azacytidinecladribine, busulfan, diethylstilbestrol, 2′,2′-difluorodeoxycytidine,docetaxel, erythrohydroxynonyladenine, ethinyl estradiol,5-fluorodeoxyuridine, 5-fluorodeoxy-uridine monophosphate, fludarabinephosphate, fluoxymesterone, flutamide, hydroxyprogesterone caproate,idarubicin, interferon, medroxyprogesterone acetate, megestrol acetate,melphalan, mitotane, paclitaxel, pentostatin,N-phosphonoacetyl-L-aspartate (PALA), plicamycin, semustine, teniposide,testosterone propionate, thiotepa, trimethylmelamine, uridine,vinorelbine, xaliplatin, gemcitabine, capecitabine, epothilone and itsnatural or synthetic derivatives, temozolomide, tositumomab (Bexxar),trabedectin, and the inhibitors of the kinesin spindle protein Eg5.

The composition of the present invention can be combined with anticanceragents, including but not limited to Tyrosine Kinase Inhibitors (TKI),as well as signal transduction inhibitors which target the EGFR familyand their related ligands including, but not limited to Herceptin(trastuzumab), Erbitux (cetuximab), pertuzumab, ZD-1839/Iressa,OSI-774/Tarceva, CI-1033, GW-2016, CP-724,714, HKI-272, and EKB-569.

In one embodiment, the composition of the present invention can becombined with inhibitors of the Raf/MEK/ERK transduction pathwayincluding, but not limited to PD-325901, and ARRY-142886.

In one embodiment, the composition of the present invention can becombined with inhibitors targeting the protein site of Rho-associatedprotein kinase 2 (ROCK2) including, but not limited to Lapatinib,Paroxetine, Rucaparib, Doxepin, Levobunolol, Darifenacin, Risperidone,Frovatriptan, Rotigotine, Rotigotine, Carteolol, Ipratropium,Testosterone, Danazol, and derivatives thereof.

In one embodiment, the composition need of the present invention can becombined with inhibitors targeting the protein site of Aurora Aincluding, but not limited to Ziprasidone, Methyltestosterone,Exemestane, Panobinostat, Dolasetron, Letrozole, Lapatinib, Lenvatinib,Losartan, Axitinib, Flavoxate, Irbesartan, Nintedanib, Dibucaine,Iloperidone, Erlotinib, Paliperidone, Alprazolam, Halcion, Regorafenib,and derivatives thereof.

In one embodiment, the composition of the present invention can becombined with inhibitors targeting the protein site ofBromodomain-containing protein 4 (BRD4) including, but not limited toNicardipine, Alfentanil, cilostazol, Alfuzosin, Pomalyst, Vemurafenib,Losartan, Salmeterol, Nebivolol, Podofilox, Midazolam, and derivativesthereof.

In one embodiment, the composition of the present invention can becombined with proteasome inhibitors and mTOR inhibitors including, butnot limited to bortezomib and CCI-779.

In one embodiment, the composition of the present invention can becombined with biological therapies including, but not limited toimmunotherapy (such as vaccines, cytokines, and some antibodies), genetherapy, and some targeted therapies.

In one embodiment, the composition of the present invention can becombined with cytokines including, but not limited to IL-2 or aninterferon (IFN), and optionally the interferon is an alpha-IFN or agamma-IFN; and optionally the IL-2 is a recombinant IL-2.

In one embodiment, the composition of the present invention can becombined with monoclonal antibody against GD2 protein including, but notlimited to Hu3F8, hu14.18K322A, Hu14.18-IL-2, and dinutuximab (Qarziba).

In embodiments, the preferable dosage of rilpivirine is from about 0.1mg to 1000 mg, and more preferably from 1 mg to 25 mg. For example, itmay be administered to the subject at a dosage of 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25mg per day.

Another aspect of the present invention provides a method for preventinggrowth of and/or treating cancerous tumors and/or delaying onset ofcancer from tumor-initiating cells, comprising administering acomposition comprising an effective amount of the compound of Formula(I) or Formula (II):

or any pharmaceutically acceptable salt thereof, to a subject in needthereof.

In an embodiment, the composition may be administered alone or incombination with one or more chemotherapeutic, biological, and/oranticancer agents to the subject in need thereof.

In an embodiment, such a method is contemplated wherein the one or morechemotherapeutic agents comprise one or more alkylating agents,anti-metabolites, antitumor antibiotics, topoisomerase inhibitors,mitotic inhibitors, steroids, and/or any mixtures thereof; and the onemore biological agents comprise one or more of vaccines, cytokines,antibodies, protein and peptide drugs and/or any mixtures thereof.

In an embodiment, the one or more alkylating agents for the method areone or more selected from cyclophosphamide, melphalan, temozolomide,carboplatin, cisplatin, and/or oxaliplatin.

In an embodiment, the one or more anti-metabolites are one or moreselected from 5-fluorouracil, 6-mercaptopurine, cytarabine, gemcitabine,and/or methotrexate.

In an embodiment, the one or more antitumor antibiotics are one or moreselected from actinomycin-D, bleomycin, daunorubicin, and/ordoxorubicin.

In an embodiment, the one or more topoisomerase inhibitors are one ormore selected from etoposide, irinotecan, teniposide, and/or topotecan.

In an embodiment, the one or more mitotic inhibitors are one or moreselected from docetaxel, estramustine, paclitaxel, and/or vinblastine.

In an embodiment, the one or more steroids are one or more selected fromprednisone, methylprednisolone, and/or dexamethasone.

In an embodiment, the one or more antibodies are selected from Hu3F8,hu14.18K322A, Hu14.18-IL-2, dinutuximab, or any combination thereof.

In an exemplary embodiment, the compound of Formula (I) or (II) and/orthe pharmaceutically acceptable salt thereof, in the composition targetstumor-initiating cells capable of growing or developing intoneuroblastoma in the subject.

As used herein, the term “prevent” or “prevention” in the context oftreatment, for example, as in “preventing cancer” or “preventing thegrowth of cancer” refers to a reduction in cancer. Prevention does notrequire 100% elimination of the symptom.

In one embodiment, said composition is formulated into one or more ofthe following administrative forms: a parenteral formulation, an aqueoussolution, a liposome, an injectable solution, suspension or emulsion, anintravenous solution, a tablet, a pill, a lozenge, a capsule, a caplet,a patch, a spray, an inhalant, a powder, a freeze-dried powder, aninhalant, a patch, a gel, a geltab, a nanosuspension, a nanoparticle, ananoliposome, a microgel, a pellet, a suppository, an oral suspension,an oral disintegrating tablet, a dispersible tablet, an oraldisintegrating film, a microemulsion, a nanoemulsion, and aself-emulsifying drug delivery system, and/or any combination thereof.

In embodiments, said cancerous tumor or cancer comprises a mastocytomaor a mast cell tumor, an ovarian cancer, a non-small cell lung cancer,small cell lung cancer, hepatocarcinoma, melanoma, retinoblastoma,breast tumor, colorectal carcinoma, leukemia, lymphoma, acutelymphoblastic leukemia (ALL) or acute lymphoid leukemia, acute myeloidleukemia (AML), a histiocytic sarcoma, a brain tumor, an astrocytoma, aglioblastoma, a neuroma, a neuroblastoma, a colon carcinoma, cervicalcarcinoma, sarcoma, prostate tumor, bladder tumor, tumor of thereticuloendothelial tissues, Wilm's tumor, ovarian carcinoma, a bonecancer, an osteosarcoma, a renal cancer, or head and neck cancer, oralcancer, a laryngeal cancer, or an oropharyngeal cancer.

In a preferred embodiment, said cancerous tumor or cancer comprisesneuroblastoma. In an exemplary embodiment, the cancerous tumor or canceris developed from neuroblastoma-initiating cells in the subject. Inother words, the compounds of Formula (I) or (II) and/or thepharmaceutically acceptable salt thereof in the present composition isfor targeting and inhibiting the proliferation of tumor-initiating cellswhich are capable of developing into neuroblastoma in the subject.

In embodiments, said subject is human comprising adult, juvenile,children and infants. In an exemplary embodiment, the subject may be onehaving neuroblastoma, diagnosed with neuroblastoma, at recognized riskof having neuroblastoma, or in recognized need of neuroblastomatreatment. In embodiments, the subject may be one having or diagnosedwith one of the other cancers contemplated herein, or at recognized riskof having one of the other cancers contemplated herein or in recognizedneed of cancer treatment.

In embodiments, the composition is administered intravenously,parenterally, intramuscularly, subcutaneously, nasally, pulmonarily,topically or locally, orally, buccally, sublingually, vaginally,rectally, instillation, injection, implantation into the eyes, surgicalimplantation, or by liposome, implant or via vessel-targetednanosuspension delivery to said subject.

In embodiments, the effective amount of said compound in the compositionis from about 0.1 mg to 1000 mg per day, and more preferably theeffective amount of said compound in the composition is from 0.1 mg to25 mg per day. For example, the compound in the composition isadministered to the subject in an effective amount of 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or25 mg per day.

In embodiments, the composition is administered at least once daily, forexample, once per day, twice per day, three times per day, four timesper day, etc.

The candidates for each of the agents described herein should beunderstood to be applicable to or combinable with the presentcomposition in any of the aspects of the present invention.

Another embodiment relates to a method for reducing the frequency oramount of a chemotherapeutic agent needed to treat cancerous tumorsand/or delay progression of cancer from tumor-initiating cells, themethod comprising administering a composition comprising an effectiveamount of a compound of Formula (I) or Formula (II):

-   -   or any pharmaceutically acceptable salt thereof, in combination        with one or more of chemotherapeutic agents, to a subject in        need thereof, the one or more of the chemotherapeutic agents        being selected from alkylating agents, anti-metabolites,        antitumor antibiotics, topoisomerase inhibitors, mitotic        inhibitors, steroids and/or any mixtures thereof; wherein the        frequency or amount of the chemotherapeutic agent administered        is approximately up to 75 to 90 percent less than a standard        protocol without administration of the composition comprising an        effective amount of the compound of Formula (I) or Formula (II).

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the present invention are described in more detailhereinafter with reference to the drawings.

FIG. 1A, FIG. 1B, and FIG. 1C illustrate the inhibition of theneuroblastoma cells IMR-32 under the treatment of rilpivirine alone;FIG. 1A is the control group and shows IMR-32 cells incubated for 24 hwithout drug treatment; FIG. 1B shows IMR-32 cells treated withrilpivirine (5 μM) for 24 h FIG. 1C shows the percentage cell viabilityof TMR-32 treated (72 h) with various concentrations of rilpivirine.

FIG. 2 presents the isobologram analysis of the combination treatment ofdoxorubicin (DOX) and rilpivirine (RPV) against IMR-32 cell line.

FIG. 3 presents the isobologram analysis of the combination treatment ofdoxorubicin (DOX) and rilpivirine (RPV) against SK-N-SH cell line.

FIG. 4 presents the isobologram analysis of the combination treatment ofdoxorubicin (DOX) and rilpivirine (RPV) against SK-N-BE(2) cell line.

FIG. 5 presents the isobologram analysis of the combination treatment ofdoxorubicin (DOX) and rilpivirine (RPV) against SH-SY5Y cell line.

FIG. 6 presents the isobologram analysis of the combination treatment of4-hydroperoxycyclophosphamide (CPA-OOH) and rilpivirine (RPV) againstIMR-32 cell line.

FIG. 7 presents the isobologram analysis of the combination treatment of4-hydroperoxycyclophosphamide (CPA-OOH) and rilpivirine (RPV) againstSH-SY5Y cell line.

FIG. 8 presents the isobologram analysis of the combination treatment of4-hydroperoxycyclophosphamide (CPA-OOH) and rilpivirine (RPV) againstSK-N-SH cell line.

FIG. 9 presents the isobologram analysis of the combination treatment of4-hydroperoxycyclophosphamide (CPA-OOH) and rilpivirine (RPV) againstSK-N-BE(2) cell line.

FIG. 10 presents the isobologram analysis of the combination treatmentcisplatin (CDDP) and of rilpivirine (RPV) against TMR-32 cell line.

FIG. 11 presents the isobologram analysis of the combination treatmentcisplatin (CDDP) and of rilpivirine (RPV) against SH-SY5Y cell line.

FIG. 12 presents the isobologram analysis of the combination treatmentcisplatin (CDDP) and of rilpivirine (RPV) against SK-N-SH cell line.

FIG. 13 presents the isobologram analysis of the combination treatmentcisplatin (CDDP) and of rilpivirine (RPV) against SK-N-BE(2) cell line.

FIG. 14 presents the isobologram analysis of the combination treatmentetoposide (ETOP) and of rilpivirine (RPV) against SH-SY5Y cell line.

FIG. 15 presents the isobologram analysis of the combination treatmentetoposide (ETOP) and of rilpivirine (RPV) against SK-N-SH cell line.

FIG. 16 presents the isobologram analysis of the combination treatmentetoposide (ETOP) and of rilpivirine (RPV) against TMR-32 cell line.

FIG. 17 presents the isobologram analysis of the combination treatmentetoposide (ETOP) and of rilpivirine (RPV) against SK-N-BE(2) cell line.

FIG. 18 presents the isobologram analysis of the combination treatmentvincristine (Vin) and of rilpivirine (RPV) against SH-SY5Y cell line.

FIG. 19 presents the isobologram analysis of the combination treatmentvincristine (Vin) and of rilpivirine (RPV) against TMR-32 cell line.

FIG. 20 presents the isobologram analysis of the combination treatmentvincristine (Vin) and of rilpivirine (RPV) against SK-N-BE(2) cell line.

FIG. 21 presents the isobologram analysis of the combination treatmentvincristine (Vin) and of rilpivirine (RPV) against SK-N-SH cell line.

FIG. 22 presents the isobologram analysis of the combination treatmentcarboplatin (CBP) and of rilpivirine (RPV) against TMR-32 cell line.

FIG. 23 presents the isobologram analysis of the combination treatmentcarboplatin (CBP) and of rilpivirine (RPV) against SH-SY5Y cell line.

FIG. 24 presents the isobologram analysis of the combination treatmentcarboplatin (CBP) and of rilpivirine (RPV) against SK-N-SH cell line.

FIG. 25 presents the isobologram analysis of the combination treatmentcarboplatin (CBP) and of rilpivirine (RPV) against SK-N-BE(2) cell line.

FIG. 26 illustrates the expressions of cleaved PARP and p-H2A.X inneuroblastoma cell lines SK-N-SH, SK-N-BE(2) and SH-SY5Y after applyingdifferent concentrations of rilpivirine.

FIG. 27 illustrates the expressions of cleaved PARP, p-H2A.X and BRCA1in neuroblastoma cell lines IMR-32 after applying differentconcentrations of rilpivirine, vincristine and rilpivirine plusvincristine.

FIG. 28 illustrates volume changes of the neuroblastoma tumor after theadministration of the cisplatin and cisplatin with rilpivirine in ananimal model.

FIG. 29 illustrates volume changes of the neuroblastoma tumor after theadministration of the carboplatin and carboplatin with rilpivirine in ananimal model.

FIG. 30 illustrates volume changes of the neuroblastoma tumor after theadministration of the vincristine and vincristine with rilpivirine in ananimal model.

FIG. 31 illustrates volume changes of the neuroblastoma tumor after theadministration of the vincristine alone and vincristine with differentconcentrations of rilpivirine (60 mg and 160 mg respectively) in ananimal model.

FIG. 32 illustrates volume changes of the neuroblastoma tumor after theadministration of rilpivirine only in an animal model.

DETAILED DESCRIPTION

The present invention relates to the use of rilpivirine, its base form,or salts thereof, alone or in combination with other cancer therapies,to treat cancer, while minimizing serious side effects from those othercancer therapies. Prevention of the genesis of cancer, as well as thesubstantial reduction or elimination of malignant cells and/or symptomsassociated with the development and metastasis of malignancies arecontemplated. The treatment of precancerous conditions is alsocontemplated herein.

Compounds of Formula I or II, shown below, may be employed in thepresent invention:

The compounds of Formula II are generally referred to as a “base” formof rilpivirine. Pharmaceutically acceptable salts of the Formula IIcompound may also be used, including, but not limited to, phosphates,acetates, maleates, and sulfates thereof. The forms may be crystallineor amorphous.

In the present invention, rilpivirine of Formula (I), (II), or its saltsis used alone or is combined with various anti-cancer agents, biologicalagents, or other treatments in order to treat a variety of cancers. Theaddition of rilpivirine to a chemotherapy regime or chemotherapeuticagent permits a substantial reduction in the amount of thechemotherapeutic agent needed to reduce tumor sizes, thereby greatlydiminishing the harmful side-effects of those chemotherapeutic agents.In some aspects, the amount of chemotherapeutic agent or biologic agentreduction may be up to 75 to 90 percent of the amount and or frequencyof the standard treatment regimen.

In one aspect, the invention includes a method for preventing growth ofand/or treating cancerous tumor and/or delaying onset of cancer fromtumor-initiating cells, by administering a composition comprising aneffective amount of a compound of Formula (I) or Formula (II):

Alternatively, a pharmaceutically acceptable salt of the compound ofFormula (II) may be used. The compound of Formula (I) or Formula (II) isadministered alone or in combination with one or more chemotherapeutic,biological and/or anticancer agents. By “in combination,” it is meantthat the composition of Formula (I) or Formula (II) may be administeredseparately, at the same time in synchrony, or by chrono-dosing,concurrent infusion or separate infusion with the one or more of thechemotherapeutic, biological and/or anticancer agents, and wherein oneof the chemotherapeutic, biological and/or anticancer agents can beadministered before or after the other.

The composition may be administered intravenously, parenterally,nasally, topically or locally, orally, or by liposome, implant or viavessel-targeted nanosuspension delivery to the subject.

While not being bound by theory, it is believed that the compounds ofFormula (I) or (II) may disrupt cancer cell replication by interferingwith DNA replication. Thus, rilpivirine may increase DNA damage, reducethe DNA repair mechanism, degrade MYCN, and/or inhibit angiogenesis.Further, the compounds of Formula (I) or (II) may shut down survivalsignals by kinase inhibition. Alternatively, the compounds of Formula(I) or (II) may increase the uptake by the targeted tumor cells ofchemotherapeutic drugs by interfering with the efflux transporters sucha p-glycoprotein and BCRP.

The chemotherapeutic agent may be one or more of alkylating agents,anti-metabolites, antitumor antibiotics, topoisomerase inhibitors,mitotic inhibitors, steroids and/or any mixtures thereof

The alkylating agents may be one or more of cyclophosphamide, melphalan,temozolomide, carboplatin, cisplatin, and/or oxaliplatin. Theanti-metabolites may be one or more of from 5-fluorouracil,6-mercaptopurine, cytarabine, gemcitabine, and/or methotrexate. Theantitumor antibiotics may be one or more of actinomycin-D, bleomycin,daunorubicin, and/or doxorubicin. The topoisomerase inhibitors may beone or more of etoposide, irinotecan, teniposide, and/or topotecan. Themitotic inhibitors may be one or more of docetaxel, estramustine,paclitaxel, and/or vinblastine. The steroids may be one or more ofprednisone, methylprednisolone, and/or dexamethasone.

The method of the present invention may be used to treat a variety ofcancers. In one aspect a cancerous tumor or cancer may be a mastocytomaor a mast cell tumor, an ovarian cancer, pancreatic cancer, a non-smallcell lung cancer, small cell lung cancer, hepatocarcinoma, melanoma,retinoblastoma, breast tumor, colorectal carcinoma, leukemia, lymphoma,acute lymphoblastic leukemia (ALL) or acute lymphoid leukemia, acutemyeloid leukemia (AML), a histiocytic sarcoma, a brain tumor, anastrocytoma, a glioblastoma, a neuroma, a neuroblastoma, a coloncarcinoma, cervical carcinoma, sarcoma, prostate tumor, bladder tumor,tumor of the reticuloendothelial tissues, Wilm's tumor, ovariancarcinoma, a bone cancer, an osteosarcoma, a renal cancer, or head andneck cancer, oral cancer, a laryngeal cancer, or an oropharyngealcancer.

The treatment of the present invention is intended to apply to humansincluding adults, juveniles, children and infants.

An effective amount of the compound in the composition is from about 0.1mg to 1000 mg per day. As is understood by those in the art, the dosingmay vary based on the type of cancer, the stage of the cancer, themethod of administration, and the amount and type of the co-administeredchemotherapeutic or biological agent. In another aspect, the range ofthe compound amount is from 0.1 mg to 25 mg per day. For example, thecompound in the composition is administered to the subject in aneffective amount of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, or 25 mg per day.

The administration time period may be one or more times per day, severaltimes per week, or once per week or multiple weeks, depending on othertreatments being performed and the type and stage of the cancer.

The biological agent may be one or more of vaccines, cytokines,antibodies, protein and peptide drugs and/or any mixtures thereof. Theantibodies may be one or more of Hu3F8, hu14.18K322A, Hu14.18-IL-2,dinutuximab or any combination thereof. In one aspect two or morechemotherapeutic, biological and/or other anticancer agents areformulated with said compound of Formula (I) or (II) for administrationto a subject.

The compound of Formula (I) or (II) may be formulated into one or moreof the following administrative forms: a parenteral formulation, anaqueous solution, a liposome, an injectable solution, an injectablesuspension, an injectable emulsion, an intravenous solution, anintravenous suspension/nanosuspension, a tablet, a pill, a lozenge, acapsule, a caplet, a patch, a spray, an inhalant, a powder, afreeze-dried powder, a patch, a gel, a geltab, a suspension, ananosuspension, a microparticle, a nanoparticle, a nanoliposome, amicrogel, a pellet, a suppository, an oral suspension, an oraldisintegrating tablet, an oral dispersible tablet, an oraldisintegrating film, a microemulsion, a nanoemulsion and aself-emulsifying drug delivery system and/or any combination thereof

Initially, isobologram and combination index analysis were performed toevaluate the drug-drug interaction between rilpivirine (RPV) and otherpotential chemotherapeutic agents. Isobologram analysis evaluates theinteraction between two drugs at a given effect level. The drug-druginteraction between rilpivirine and other chemotherapeutic agents wasanalyzed against neuroblastoma cell lines such as CHP-100, CHP-126,CHP-134B, CHP-212, CHP-234, CHP-382, CHP-404 (Schlesinger et al., 1976),GI-CA-N (Donti et al., 1988), GI-LI-N (Cornaglia et al., 1992), GI-ME-N(Ponzoni et al., 1988), GOTO (Sekiguichi et al., 1979), IGR-N-835(Bettan et al., 1989), TMR-32 (Tumilocwicz et al., 1970), LA-N-1, LA-N-5(Seeger et al., 1977), MHH-NB11 (Pietsch et al., 1988), NB-69 (Gilbertet al., 1982), NB1-G (Carachi et al., 1987), NBL-W (Foley et al., 1991),NGP, NGP-2, NLF, NMB (Brodeur et al., 1977), RN-GA (Scarpa et al.,1989), SK-N-AS, SK-N-DZ, SK-N-FI, SK-N-LE, SK-PN-LO, SK-PN-LI, SK-PN-DW,VA-N-BR (Helson and Helson, 1985), SK-N-BE(2) (Biedler and Spengler,1976), SK-N-SH (Biedler et al., 1983), SMS-KAN, SMS-KANR, SMS-KCN,SMS-KCNR (Reynolds et al., 1986), TC-32, TC-106, N1000, N1008, N1016,A4573 (Whang-Peng et al., 1986), LAP-35 (Bagnara et al., 1990), NUB-20(Yeger et al., 1990), SK-N-MC (Biedler et al., 1983), and TC-268(Cavazzana et al, 1988); all of which of incorporated herein byreference in their entireties.

Neuroblastoma Cell Lines

IMR-32 cell line is a human neuroblastoma cell line established from anabdominal mass in a 13-month-old Caucasian male (Tumilocwicz et al.,1970; Rostomily R C, et al., 1997; Maestrini E, et al. 1996), and hasbeen widely used as a model for neural related disease. In addition toIMR-32 cell line, the growth of three additional neuroblastoma celllines (SK-N-BE(2), SK-N-SH, and SH-SY5Y) were also successfullyinhibited under rilpivirine treatment.

Isobologram Analysis

In the present invention, the IC₅₀ concentrations required to inhibitthe activity of IMR-32 cell line and other neuroblastoma cell lines weredetermined for rilpivirine and other chemotherapeutic agents, andpresented on the two-coordinate plot with x- and y-axes, forming the twopoints (IC_(50, rilpivirine), 0) and (0,IC_(50, chemotherapeutic agents)). The line connecting these two pointsshows the additivity of these two drugs. Moreover, the concentration orratios of rilpivirine in combination with chemotherapeutic agents whichprovide the same inhibition effect are denoted as point(C_(rilpivirine, 50), C_(chemotherapeutic agents, 50)) in the same plot.When this point is located below, on, or above the line, it showssynergy, additivity, and antagonism, respectively.

Combination Index (CI) Analysis

The combination index provides a quantitative measurement of thedrug-drug interaction at the given effect level. The combination index(CI) is calculated by following equation:

${CI} = {\frac{{{IC}_{50}(A)}_{pair}}{{IC}_{50}(A)} + \frac{{{IC}_{50}(B)}_{pair}}{{IC}_{50}(B)}}$

where “IC₅₀(A)_(pair)” refers to IC₅₀ of drug A when used in combinationwith drug B, “IC₅₀(B)_(pair)” refers to IC₅₀ of drug B when used incombination with drug A, “IC₅₀(A)” refers to IC₅₀ of drug A when it isused alone, and “IC₅₀(B)” refers to IC₅₀ of drug B when it is usedalone.

A CI of less than, equal to, and more than 1 indicates synergistic,additive, and antagonistic effect, respectively.

Xenograft Mouse Model

This study employed female BALB/c nude mice, aged 4-5 weeks, obtainedfrom BioLasco Taiwan (under Charles River Laboratories Licensee). Theanimals were housed in individually ventilated cages (IVC, 36 MiniIsolator system). The allocation for 4 animals was 27×20×14 in cm. Allthe animals were maintained in a hygienic environment under controlledtemperature (20-24° C.) and humidity (30%-70%) with 12-hour light/darkcycle. Free access to standard lab diet [MFG (Oriental Yeast Co., Ltd.,Japan)] and autoclaved tap water were granted. All aspects of this workincluding housing, experimentation, and animal disposal were performedin general accordance with the “Guide for the Care and Use of LaboratoryAnimals: Eighth Edition” (National Academies Press, Washington, D.C.,2011) in our AAALAC-accredited laboratory animal facility. In addition,the animal care and use protocol was reviewed and approved by the IACUCat Pharmacology Discovery Services, Taiwan.

The IMR-32 tumor cell line was purchased from American Type CultureCollection (ATCC CCL-127, neuroblastoma) and cultured in PharmacologyDiscovery Services, Taiwan. The cells were cultured in MEM mediumcontaining 10% fetal bovine serum (FBS), 1 mM sodium pyruvate and 1 mMNEAA at 37° C. in 5% CO2 incubator. Female BALB/c nude mice were used asdescribed in the preceding section. Viable IMR-32 cells (ATCC CCL-127)were subcutaneously (SC) implanted (1×10⁷ cells in 1:1 matrigel/completemedia mixture at 0.2 mL/mouse) into the right flank of female BALB/cnude mice. Forty-one days post tumor cell implantation (group mean tumorvolumes ranging 128 mm³-130 mm³), all the animals were randomized intosixteen study groups, each containing eight animals, and doseadministrations were initiated (denoted as Day 1). All the experimentswere conducted using protocols and conditions approved by theinstitutional animal care and use committee. The tumor volume, bodyweight, mortality, and signs of overt toxicity were monitored andrecorded twice weekly for 28 days.

Cell Culture and Drug Treatment

The IMR-32, SK-N-SH, SH-SY5Y and SK-N-BE(2) tumor cell lines werepurchased from American Type Culture Collection (ATCC, Manassas, Va.).IMR-32 and SK-N-SH were cultured in EMEM medium containing 10% fetalbovine serum (FBS), 100 U/mL penicillin and 100 U/mL streptomycin at 37°C. in 5% CO2 incubator. SH-SY5Y and SK-N-BE(2) were cultured inEMEM/F-12 containing 10% FBS, 100 U/mL penicillin and 100 U/mLstreptomycin at 37° C. in 5% CO2 incubator. Cells were plated in 96 wellmicroplates at a 3×10³ cells density per well in culture medium. After24 hours cells were treated with different concentrations of rilpivirineand chemotherapy agents. The cells were incubated for 72 hours and thencell proliferation was measured by3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT)colorimetric assays (Sigma-Aldrich, Inc., St. Louis, Mo.). The MTTassays were performed in triplicate. Absorbance of each well wasmeasured with a spectrophotometer (uQuant SpectroMAX GeminiDual-scanning microplate spectro, Bio-tek Instrument Inc.) at 550 nm.Growth inhibition was expressed as the ratio of the mean absorbance oftreated cells relative to that of the control.

Tumor Volume Evaluation

Tumor volume (mm³) was estimated according to the prolate ellipsoidformula as:

Length×(Width)²×0.5.

Tumor growth inhibition (T/C) was calculated by the following formula:

% T/C=(T _(n) /C _(n))×100%

C_(n): Tumor volume measured on Day n in the control group

T_(n): Tumor volume measured on Day n in the treated group

Percent tumor growth inhibition (TGI) was also calculated by thefollowing formula:

% TGI=(1−(T _(n) /C _(n)))×100%

Two-way ANOVA followed by Bonferroni test was also used to ascertain thestatistically significant difference in anti-tumor activity compared tothe negative control group in the study (*p<0.05).

An in vivo study was performed to determine the influence of rilpivirineon rapid COJEC (cisplatin [C], vincristine [O], carboplatin [J],etoposide [E], and cyclophosphamide [C]), one of the standard regimensfor the treatment of high-risk neuroblastoma patients. Rilpivirine wascombined with each from rapid COJEC and assessed for the potentialbenefit in in the treatment of neuroblastoma in a xenograft mouse model.As will be seen in Example 4 below, rilpivirine was determined topotentiate the therapeutic effects of cisplatin, carboplatin andvincristine. By potentiating the therapeutic effect of thesechemotherapeutic agents, the dosing frequency or drug concentration ofthe cytotoxic chemotherapy drugs during the treatment of neuroblastomamay be reduced, particularly, they may be reduced up to 75 to 90 percentof the amount of standard therapeutic protocols. Further, rilpivirinedoes not weaken the therapeutic effects of the chemotherapy drugs(cyclophosphamide and etoposide), showing that it can be used with thestandard treatment regimen for neuroblastoma.

In Vitro Analysis

Example 1 (In Vitro Data of Rilpivirine Alone Against MultipleNeuroblastoma Cell Lines)

To determine the potency of rilpivirine for the inhibition ofneuroblastoma cell lines, IC₅₀ was measured in neuroblastoma cell linessuch as IMR-32, SK-N-BE(2), SK-N-SH, and SH-SY5Y (Table 1). Further, thegrowth of neuroblastoma cell line TMR-32 was significantly inhibitedunder 5 μM rilpivirine treatment for 24 hours (FIG. 1A, FIG. 1B).Percentage cell viability of IMR-32 treated with various concentrationsof rilpivirine is shown in FIG. 1C.

TABLE 1 IC₅₀ values of rilpivirine alone in neuroblastoma cell linesCell lines IMR-32 SK-N-BE(2) SK-N-SH SH-SY5Y IC₅₀ (μM) 2.97 3.37 2.753.12

Example 2 (In Vitro Data of Rilpivirine in Combination with DoxorubicinMultiple Neuroblastoma Cell Lines)

Doxorubicin (DOX) is a chemotherapeutic agent for cancer treatmentincluding breast cancer, bladder cancer, Kaposi's sarcoma, lymphoma, andacute lymphocytic leukemia. Isobologram analysis shows synergisticeffects with different ratios of rilpivirine and DOX against TMR-32 andSK-N-SH cell lines (FIG. 2 and FIG. 3). Combination index analysis alsoshows synergistic effect that CI is less than 1 (Table 2 and Table 3).As for SK-N-BE(2) and SH-SY5Y cell lines, there exists additive effectsin isobologram analysis (FIG. 4 and FIG. 5) and CI is approximatelyclose to 1 (Table 4 and Table 5).

TABLE 2 Combination index of doxorubicin (DOX) to rilpivirine (RPV)combination treatment against IMR-32 cell line Drug concentration ratio[μM (micromolar)] DOX RPV Combination index 1 1.25 0.447 1 2.5 0.262 1 50.295 1 10 0.211 1 20 0.515 1 40 0.795 1 80 1.389

TABLE 3 Combination index of doxorubicin (DOX) to rilpivirine (RPV)combination treatment against SK-N-SH cell line Drug concentration ratio[μM (micromolar)] DOX RPV Combination index 1 2.5 0.282 1 1.25 0.366 10.625 0.328 1 5 0.297 1 10 0.516 1 20 0.664 1 40 0.744

TABLE 4 Combination index of doxorubicin (DOX) to rilpivirine (RPV)combination treatment against SK-N-BE(2) cell line Drug concentrationratio [μM (micromolar)] DOX RPV Combination index 1 2.5 1.100 1 1.250.879 1 0.625 0.781 1 5 1.030 1 10 1.259 1 20 1.357 1 40 1.175

TABLE 5 Combination index of doxorubicin (DOX) to rilpivirine (RPV)combination treatment against SH-SY5Y cell line Drug concentration ratio[μM (micromolar)] DOX RPV Combination index 1 2.5 0.683 1 1.25 0.782 10.625 1.023 1 5 0.695 1 10 0.751 1 20 0.887 1 40 0.835

Example 3 (In Vitro Data of Rilpivirine in Combination with PerfosfamideAgainst Multiple Neuroblastoma Cell Lines)

4-hydroperoxycyclophosphamide (or perfosfamide), a cyclophosphamideanalog, was an experimental drug for blood cancer treatment such asacute myeloid leukemia. The isobologram analysis shows additive and mildantagonistic effects with different ratios of4-hydroperoxycyclophosphamide and rilpivirine against IMR-32 (FIG. 6),SH-SY5Y (FIG. 7), SK-N-SH (FIG. 8) and SK-N-BE(2) cell lines (FIG. 9).The combination index analysis also shows additive that CI is between0.9 to 1.1 and antagonistic effect that CI is more than 1.1 (Table 6 toTable 9).

TABLE 6 Combination index of 4-hydroperoxycyclophosphamide (CPA-OOH) torilpivirine (RPV) combination treatment against IMR-32 cell line Drugconcentration ratio [μM (micromolar)] CPA-OOH RPV Combination index 1 81.360 1 4 1.217 1 2 1.091 1 1 1.047 2 1 0.813 4 1 0.844 8 1 0.929

TABLE 7 Combination index of 4-hydroperoxycyclophosphamide (CPA-OOH) torilpivirine (RPV) combination treatment against SH-SY5Y cell line Drugconcentration ratio [μM (micromolar)] CPA-OOH RPV Combination index 1 81.036 1 4 1.057 1 2 1.190 1 1 1.026 2 1 0.989 4 1 0.956 8 1 1.033

TABLE 8 Combination index of 4-hydroperoxycyclophosphamide (CPA-OOH) torilpivirine (RPV) combination treatment against SK-N-SH cell line Drugconcentration ratio [μM (micromolar)] CPA-OOH RPV Combination index 1 81.079 1 4 0.969 1 2 1.178 1 1 1.083 2 1 0.880 4 1 1.178 8 1 1.094

TABLE 9 Combination index of 4-hydroperoxycyclophosphamide (CPA-OOH) torilpivirine (RPV) combination treatment against SK-N-BE(2) cell lineDrug concentration ratio [μM (micromolar)] CPA-OOH RPV Combination index1 8 0.973 1 4 0.913 1 2 0.904 1 1 0.951 2 1 0.752 4 1 0.778 8 1 1.010

Example 4 (In Vitro Data of Rilpivirine in Combination with CisplatinAgainst Multiple Neuroblastoma Cell Lines)

Cisplatin (CDDP) is a chemotherapeutic agent for cancer treatmentincluding testicular cancer, ovarian cancer, cervical cancer, breastcancer, bladder cancer, head and neck cancer, esophageal cancer, lungcancer, mesothelioma, brain tumors and neuroblastoma. Isobologramanalysis shows additive and mild antagonistic effects with differentratios of rilpivirine and CDDP against IMR-32 (FIG. 10), SH-SY5Y (FIG.11), SK-N-SH (FIG. 12), and SK-N-BE(2) cell lines (FIG. 13). Thecombination index analysis also shows additive effect that CI is between0.9 and 1.1 and antagonistic effect that CI is more than 1.1 (Table 10to Table 13).

TABLE 10 Combination index of cisplatin (CDDP) to rilpivirine (RPV)combination treatment against IMR-32 cell line Drug concentration ratio[μM (micromolar)] Combination Index CDDP RPV 8 1 1.219 4 1 1.117 2 11.086 1 1 1.183 1 2 1.056 1 4 1.081 1 8 1.164

TABLE 11 Combination index of cisplatin (CDDP) to rilpivirine (RPV)combination treatment against SH-SY5Y cell line Drug concentration ratio[μM (micromolar)] CDDP RPV Combination Index 8 1 1.069 4 1 1.082 2 11.239 1 1 1.305 1 2 1.255 1 4 1.088 1 8 0.997

TABLE 12 Combination index of cisplatin (CDDP) to rilpivirine (RPV)combination treatment against SK-N-SH cell line Drug concentration ratio[μM (micromolar)] Combination CDDP RPV Index 8 1 1.021 4 1 1.255 2 10.865 1 1 1.270 1 2 1.239 1 4 1.153 1 8 1.078

TABLE 13 Combination index of cisplatin (CDDP) to rilpivirine (RPV)combination treatment against SK-N-BE(2) cell line Drug concentrationratio [μM (micromolar)] Combination CDDP RPV Index 8 1 1.159 4 1 1.074 21 0.977 1 1 0.955 1 2 0.901 1 4 0.901 1 8 0.942

Example 5 (In Vitro Data of Rilpivirine in Combination with EtoposideAgainst Multiple Neuroblastoma Cell Lines)

Etoposide (ETOP) is a chemotherapy medication used for cancer treatmentincluding testicular cancer, lung cancer, lymphoma, leukemia,neuroblastoma, and ovarian cancer. Isobologram analysis showssynergistic effects with different ratios of rilpivirine and ETOPagainst SH-SY5Y and SK-N-SH cell lines (FIG. 14 and FIG. 15) and thecombination index analysis also shows synergistic effect that the CI isless than 1 (Table 14 and Table 15). As for TMR-32 cell line andSK-N-BE(2) cell line, there exists additive effects in isobologramanalysis (FIG. 16 and FIG. 17) and CI is approximately close to 1 (Table16 and Table 17).

TABLE 14 Combination index of etoposide (ETOP) to rilpivirine (RPV)combination treatment against SH-SY5Y cell line Drug concentration ratio[μM (micromolar)] Combination ETOP RPV Index 8 1 0.925 4 1 0.779 2 10.657 1 1 0.673 1 2 0.615 1 4 0.515 1 8 0.567

TABLE 15 Combination index of etoposide (ETOP) to rilpivirine (RPV)combination treatment against SK-N-SH cell line Drug concentration ratio[μM (micromolar)] Combination ETOP RPV Index 8 1 1.009 4 1 1.028 2 10.901 1 1 0.891 1 2 0.824 1 4 0.651 1 8 0.587

TABLE 16 Combination index of etoposide (ETOP) to rilpivirine (RPV)combination treatment against IMR-32 cell line Drug concentration ratio[μM (micromolar)] Combination ETOP RPV Index 8 1 1.020 4 1 1.048 2 10.972 1 1 1.061 1 2 1.008 1 4 0.907 1 8 1.018

TABLE 17 Combination index of etoposide (ETOP) to rilpivirine (RPV)combination treatment against SK-N-BE(2) cell line Drug concentrationratio [μM (micromolar)] Combination ETOP RPV Index 8 1 1.033 4 1 1.071 21 0.942 1 1 1.126 1 2 0.533 1 4 0.976 1 8 1.020

Example 6 (In Vitro Data of Rilpivirine in Combination with VincristineMultiple Neuroblastoma Cell Lines)

Vincristine (Vin) is a chemotherapy medication used for cancer treatmentincluding acute lymphocytic leukemia, acute myeloid leukemia, Hodgkin'sdisease, neuroblastoma, and small cell lung cancer. Isobologram analysisshows synergistic effects with different ratios of rilpivirine and Vinagainst SH-SY5Y cell line (FIG. 18) and the combination index analysisalso shows synergistic effect that the CI is less than 1 (Table 18). Asfor TMR-32 cell line and SK-N-BE(2) cell line, there exists additivityeffects in isobologram analysis (FIG. 19 and FIG. 20) and the CI isapproximately close to 1 (Table 19 and Table 20). Further, as forSK-N-SH cell line, there exists multiple effects in differentcombination ratios in isobologram analysis (FIG. 21) and CI is between0.5 to 1.5 (Table 21).

TABLE 18 Combination index of vincristine (Vin) to rilpivirine (RPV)combination treatment against SH-SY5Y cell line Drug concentration ratio[μM (micromolar)] Combination Vin RPV Index 1 6.25 0.914 1 12.5 0.756 125 0.637 1 50 0.657 1 100 0.527 1 200 0.371 1 400 0.373

TABLE 19 Combination index of vincristine (Vin) to rilpivirine (RPV)combination treatment against IMR-32 cell line Drug concentration ratio[μM (micromolar)] Combination Vin RPV Index 1 12.5 1.014 1 25 0.965 1 500.891 1 100 0.953 1 200 0.652 1 400 0.862 1 800 0.969

TABLE 20 Combination index of vincristine (Vin) to rilpivirine (RPV)combination treatment against SK-N-BE(2) cell line Drug concentrationratio [μM (micromolar)] Combination Vin RPV Index 1 12.5 1.158 1 250.992 1 50 1.037 1 100 1.048 1 200 1.020 1 400 1.017 1 800 0.973

TABLE 21 Combination index of vincristine (Vin) to rilpivirine (RPV)combination treatment against SK-N-SH cell line Drug concentration ratio[μM (micromolar)] Combination Vin RPV Index 1 6.25 1.492 1 12.5 1.115 125 1.174 1 50 1.029 1 100 0.819 1 200 0.623 1 400 0.571

Example 7 (In Vitro Data of Rilpivirine in Combination with CarboplatinAgainst Multiple Neuroblastoma Cell Lines)

Carboplatin (CBP) is a chemotherapy medication used for cancer treatmentincluding ovarian cancer, lung cancer, head and neck cancer, braincancer, and neuroblastoma. The isobologram analysis shows mildsynergistic effects with different combinations of rilpivirine and Vinagainst IMR-32 cell line (FIG. 22) and the combination index analysisalso shows synergistic effect that the CI is less than 1 (Table 22). Asfor SH-SY5Y cell line and SK-N-SH cell line, it shows additivity effectsin isobologram analysis (FIG. 23 and FIG. 24) and the CI isapproximately close to 1 (Table 23 and Table 24). Further, as forSK-N-BE(2) cell line, it shows antagonist effects in isobologramanalysis (FIG. 25) and CI is over 1.3 (Table 25).

TABLE 22 Combination index of carboplatin (CBP) to rilpivirine (RPV)combination treatment against IMR-32 cell line Drug concentration ratio[μM (micromolar)] Combination CBP RPV Index 1 2 0.918 1 1 0.874 2 10.909 4 1 0.778 8 1 0.711 16 1 0.726 32 1 1.018

TABLE 23 Combination index of carboplatin (CBP) to rilpivirine (RPV)combination treatment against SH-SY5Y cell line Drug concentration ratio[μM (micromolar)] Combination CBP RPV Index 1 2 0.950 1 1 0.957 2 11.019 4 1 1.230 8 1 1.053 16 1 1.086 32 1 1.046

TABLE 24 Combination index of carboplatin (CBP) to rilpivirine (RPV)combination treatment against SK-N-SH cell line Drug concentration ratio[μM (micromolar)] Combination CBP RPV Index 1 2 1.033 1 1 0.966 2 10.966 4 1 0.973 8 1 0.978 16 1 1.077 32 1 1.061

TABLE 25 Combination index of carboplatin (CBP) to rilpivirine (RPV)combination treatment against SK-N-BE(2) cell line Drug concentrationratio [μM (micromolar)] Combination CBP RPV Index 1 2 1.159 1 1 0.976 21 1.134 4 1 1.077 8 1 0.870 16 1 0.908 32 1 1.084

Example 8 (In Vitro Data of Rilpivirine Alone Against Other Cancer CellLines)

In addition to neuroblastoma cell lines, the potency of rilpivirinealone has also been shown in other cancer cell lines (Table 26).

TABLE 26 EC₅₀ values of each cancer cell lines applied with rilpivirinealone. Tumor sub-type Cell Line EC50 (uM) Acute lymphoblastic leukemiaMOLT-16 3.38 Acute lymphoblastic leukemia Jurkat 3.68 Acutelymphoblastic leukemia MOLT-4 3.75 Acute lymphoblastic leukemia CEM-C24.04 Acute monocytic leukemia Thp1 4.29 Acute T-cell leukemia J-RT3-T3-54.04 Biphenotypic B myelomonocytic leukemia MV-4-11 4.88 B-cellnon-Hodgkin lymphoma SU-DHL-4 4.29 B-cell precursor leukemia (CML)BV-173 3.69 Bladder 647-V 12.3 Breast T47D 3.23 Burkitt's lymphoma Daudi4.01 Burkitt's lymphoma EB2 4.81 Burkitt's lymphoma GA-10 3.70 Burkitt'slymphoma ST486 3.76 B-cell lymphoma DOHH-2 4.45 Chronic myelogenousleukemia EM-2 3.96 Chronic myelogenous leukemia KU812 4.09 ColonRKO-AS45-1 4.40 Duodenum HuTu80 3.59 Erythroleukemia TF-1 3.76Glioblastoma/malignant glioma U-87 MG 14.4 Histiocytic lymphoma TUR 2.08Histiocytic lymphoma U-937 3.37 Kidney Caki-2 4.05 Large cell lymphomaSU-DHL-8 4.19 Large cell immunoblastic lymphoma SR 3.78 Liver HUH-6 2.41Mantle cell lymphoma JVM-2 2.74 Medulloblastoma D283Med 3.54 MelanomaHMCB 3.49 Multiple myeloma IM-9 4.13 NSCLC COR-L105 3.11 NeuroblastomaCHP-212 3.26 Neuroblastoma SK-N-AS 4.84 Pancreas BxPC-3 11.3 PancreasPANC-1 8.27 Prostate PC-3 6.23 SCLC DMS273 4.96 Stomach SNU-16 4.23Uterine sarcoma MES-SA 4.91

Referring to FIG. 26, by increasing the concentrations of rilpivirine(0.12, 0.37, 1.11, 3.3, and 10 μM) applied on SK-N-SH, SK-N-BE(2) andSH-SY5Y neuroblastoma cell lines, the expression of cleaved poly(ADP-ribose) polymerase (PARP) and phosphor-histone H2A.X (p-H2A.X)increased gradually, suggesting rilpivirine alone can cause DNA damageand lead to cell apoptosis of neuroblastoma or other cancer cells.Furthermore, comparing to single drug treatments, combinations ofrilpivirine with chemotherapy drug vincristine showed increasedexpressions of cleaved PARP and P-H2A.X, suggesting an enhancement onDNA damage and cell apoptosis in neuroblastoma cell line TMR-32, inaddition, and tumor suppressor gene BRCA1 was also upregulated. (FIG.27).

In Vivo Analysis

In the present invention, rilpivirine was used alone or in combinationwith chemotherapy drugs such as cisplatin, carboplatin, and vincristine,to evaluate the treatment efficacy in a xenograft mouse model ofneuroblastoma. Please refer to the Xenograft Mouse Model section abovefor the details of the in vivo tests.

Example 9 (In Vivo Data of Rilpivirine with Cisplatin on Mouse Model)

As shown in FIG. 28, the tumor volume resulting from the combinationtherapy comprising intraperitoneal injection of cisplatin at 7 mg/kg onDay 1 and oral administration of rilpivirine at 400 mg/kg once daily for21 consecutive days was smaller than that of the monotherapy groupcomprising intraperitoneal injection of cisplatin at 7 mg/kg weekly forthree weeks, especially on Day 11, in a statistically significant manner(unpaired student's t-test, p<0.05), wherein the tumor growth inhibitionis approximately over 88%. The maximum % TGI values of approximately 94%appears to be on Day 28.

Example 10 (In Vivo Data of Rilpivirine with Carboplatin on Mouse Model)

Further, as shown in FIG. 29, the tumor volume resulting from thecombination therapy comprising intraperitoneal injection of carboplatinat 80 mg/kg on Day 1 and oral administration of rilpivirine at 400 mg/kgdaily (QD×11) was smaller than that of the monotherapy group comprisingintraperitoneal injection of carboplatin at 80 mg/kg weekly for threeweeks, especially on Day 8 and Day 11, in a statistically significantmanner (unpaired student's t-test, p<0.05), wherein the tumor growthinhibition is approximately over 77% and 84%. The maximum % TGI valuesof approximately 94% appears to be on Day 28.

Example 11 (In Vivo Data of Rilpivirine with Vincristine on Mouse Model)

Further, as shown in FIG. 30, the tumor volume resulting from thecombination therapy comprising intraperitoneal injection of vincristineat 1 mg/kg on Day 1 and oral administration of rilpivirine at 400 mg/kgdaily (QD×11) was smaller than that of the monotherapy group comprisingintraperitoneal injection of vincristine at 1 mg/kg weekly for threeweeks, especially on Day 8 and Day 11, in a statistically significantmanner (unpaired t-test, p<0.05), wherein the tumor growth inhibition isapproximately over 86%. The maximum % TGI values of approximately 93%appears to be on Day 28.

It is worth noting that the chemotherapy drugs in the combinationtherapy groups (in Examples 9-11) were applied on Day 1 only, and as forthe monotherapy groups, the chemotherapy drugs were applied on Day 1,Day 8 and Day 15. This indicates that administration of rilpivirinecould reduce the dosage frequency or amount of cytotoxic chemotherapydrugs, thus potentially ameliorating the side-effects caused by thechemotherapy drugs. The dosage frequency was approximately reduced fromthrice a month to once a month or twice a week to once a week.

In another embodiment, the intraperitoneal injection of carboplatin orvincristine combination therapy with rilpivirine, all drug treatment wasstopped after Day 11, and the tumor volume was not statisticallydifferent from the monotherapy groups, suggesting long-term inhibitioneffect of rilpivirine.

The above in vivo combination studies were performed to elucidate theinfluence of rilpivirine on rapid COJEC, one of the standard regimensfor the treatment of high-risk neuroblastoma patients. Rilpivirine wascombined with each chemotherapy drug in rapid COJEC (includes cisplatin,carboplatin, vincristine, cyclophosphamide and etoposide) and thepotential benefit was assessed in a xenograft mouse model.

Based on the data from this in vivo study, rilpivirine potentiated theefficacy of cisplatin, carboplatin and vincristine against theneuroblastoma xenograft model. Generally speaking, combination therapybrought a statistically significant reduction in the tumor volume(unpaired student's t-test, p<0.05) compared to that of the monotherapygroups on Day 8 and 11 for carboplatin and vincristine and Day 11 forcisplatin. It is worth noting that the chemotherapeutic drugs in thecombination therapy groups were dosed on Day 1 only. Meanwhile, in themonotherapy groups, the chemotherapeutic drugs were dosed on Day 1, Day8 and Day 15. This may indicate that rilpivirine could reduce the dosingfrequency of cytotoxic chemotherapeutic drugs.

In addition, rilpivirine+cisplatin group was observed to achieve similartumor size (unpaired student's t-test, p>0.05) compared with thecisplatin alone group from Day 11 to 15 and no difference until Day 25.For the carboplatin and vincristine combination groups, we stopped thetreatment on Day 11 but the tumor size was not statistically differentfrom the chemo alone groups.

On the other hand, we also observed that rilpivirine did not interfereor weaken the therapeutic effect of cyclophosphamide and etoposide inthe xenograft model. There was no significant difference in the tumorsize between the monotherapy and the combination therapy in the 28-dayin vivo studies which compared cyclophosphamide alone againstcyclophosphamide+rilpivirine and etoposide alone againstetoposide+rilpivirine, respectively.

Based on this in vivo study, rilpivirine was found to potentiate thetherapeutic effects of cisplatin, carboplatin and vincristine andpotentially reduce the dosing frequency of the cytotoxic chemotherapydrugs during the treatment of neuroblastoma. On the other hand,rilpivirine did not weaken the therapeutic effects of cyclophosphamideand etoposide. Therefore, superiority and improvement may be observed byincorporating rilpivirine with standard chemotherapy againstneuroblastoma.

Example 12 (In Vivo Data of Rilpivirine Alone on Mouse Model)

As shown in FIG. 32, compared to the negative control groupadministering vehicle (0.5% HPMC) once daily for 21 consecutive days viaoral administration, tumor growth was significantly inhibited in thegroup administering rilpivirine 400 mg/kg once daily for 21 consecutivedays via oral administration (unpaired student's t-test, p<0.05),indicating anti-tumor activity of rilpivirine alone againstneuroblastoma. During the course of the administration, rilpivirine waswell tolerated and not associated with body weight loss and no overttoxicities were observed.

References in the specification to “one embodiment”, “an embodiment”,“an example embodiment”, etc., indicate that the embodiment describedcan include a particular feature, structure, or characteristic, butevery embodiment may not necessarily include the particular feature,structure, or characteristic. Moreover, such phrases are not necessarilyreferring to the same embodiment. Further, when a particular feature,structure, or characteristic is described in connection with anembodiment, it is submitted that it is within the knowledge of oneskilled in the art to affect such feature, structure, or characteristicin connection with other embodiments whether or not explicitlydescribed.

In the methods of preparation described herein, the steps can be carriedout in any order without departing from the principles of the invention,except when a temporal or operational sequence is explicitly recited.Recitation in a claim to the effect that first a step is performed, andthen several other steps are subsequently performed, shall be taken tomean that the first step is performed before any of the other steps, butthe other steps can be performed in any suitable sequence, unless asequence is further recited within the other steps. For example, claimelements that recite “Step A, Step B, Step C, Step D, and Step E” shallbe construed to mean step A is carried out first, step E is carried outlast, and steps B, C, and D can be carried out in any sequence betweensteps A and E, and that the sequence still falls within the literalscope of the claimed process. A given step or sub-set of steps can alsobe repeated. Furthermore, specified steps can be carried outconcurrently unless explicit claim language recites that they be carriedout separately.

Definitions for selected terms used herein may be found within thedetailed description of the present invention and apply throughout.Unless otherwise defined, all other technical terms used herein have thesame meaning as commonly understood to one of ordinary skill in the artto which the present invention belongs.

It will be appreciated by those skilled in the art, in view of theseteachings, that alternative embodiments may be implemented withoutdeviating from the spirit or scope of the invention, as set forth in theappended claims. This invention is to be limited only by the followingclaims, which include all such embodiments and modifications when viewedin conjunction with the above specification and accompanying drawings.

1. A method for preventing growth of and/or treating a cancerous tumorand/or delaying onset of cancer from tumor-initiating cells, the methodcomprising: administering a composition comprising an effective amountof a compound of Formula (I) or Formula (II):

or any pharmaceutically acceptable salt thereof, to a subject in needthereof.
 2. The method of claim 1, wherein the cancerous tumor or cancercomprises a mastocytoma or a mast cell tumor, an ovarian cancer, anon-small cell lung cancer, small cell lung cancer, hepatocarcinoma,melanoma, retinoblastoma, breast tumor, colorectal carcinoma, leukemia,lymphoma, acute lymphoblastic leukemia (ALL) or acute lymphoid leukemia,acute myeloid leukemia (AML), a histiocytic sarcoma, a brain tumor, anastrocytoma, a glioblastoma, a neuroma, a neuroblastoma, a coloncarcinoma, cervical carcinoma, sarcoma, prostate tumor, bladder tumor,tumor of the reticuloendothelial tissues, Wilm's tumor, ovariancarcinoma, a bone cancer, an osteosarcoma, a renal cancer, or head andneck cancer, oral cancer, a laryngeal cancer, or an oropharyngealcancer.
 3. The method of claim 2, wherein the cancerous tumor or cancercomprises neuroblastoma.
 4. The method of claim 1, wherein said subjectis a human comprising adult, juvenile, children or infants.
 5. Themethod of claim 1, wherein the effective amount of the compound in thecomposition is from about 0.1 mg to 1000 mg per day.
 6. The method ofclaim 5, wherein the effective amount of said compound in thecomposition is from 1 mg to 25 mg per day.
 7. The method of claim 5,wherein the composition is administered at least once daily.
 8. Themethod of claim 1, wherein the administering a composition of Formula(I) is in combination with one or more chemotherapeutic agents,biological agents and/or anticancer agents, to a subject in needthereof.
 9. The method of claim 8 wherein the one or morechemotherapeutic agents comprises one or more alkylating agents,anti-metabolites, antitumor antibiotics, topoisomerase inhibitors,mitotic inhibitors, steroids, and/or any mixtures thereof; and the oneor more biological agents comprises one or more vaccines, cytokines,antibodies, protein and peptide drugs and/or any mixtures thereof. 10.The method of claim 9, wherein said alkylating agents are one or moreselected from cyclophosphamide, melphalan, temozolomide, carboplatin,cisplatin, and/or oxaliplatin.
 11. The method of claim 9, wherein saidanti-metabolites are one or more selected from 5-fluorouracil,6-mercaptopurine, cytarabine, gemcitabine, and/or methotrexate.
 12. Themethod of claim 9, wherein said antitumor antibiotics are one or moreselected from actinomycin-D, bleomycin, daunorubicin, and/ordoxorubicin.
 13. The method of claim 9, wherein said topoisomeraseinhibitors are one or more selected from etoposide, irinotecan,teniposide, and/or topotecan.
 14. The method of claim 9, wherein saidmitotic inhibitors are one or more selected from docetaxel,estramustine, paclitaxel, and/or vinblastine.
 15. The method of claim 9,wherein said steroids are one or more selected from prednisone,methylprednisolone, and/or dexamethasone.
 16. The method of claim 9,wherein said antibodies are selected from Hu3F8, hu14.18K322A,Hu14.18-IL-2, dinutuximab or any combination thereof.
 17. A compositionfor preventing growth of and/or treating a cancerous tumor and/ordelaying onset of cancer from tumor-initiating cells, the compositioncomprising: an effective amount of a compound of Formula (I) or Formula(II):

or any pharmaceutically acceptable salt thereof, the composition furtherincluding one or more of A and B: A: one or more chemotherapeutic agentsselected from alkylating agents, anti-metabolites, antitumorantibiotics, topoisomerase inhibitors, mitotic inhibitors, or steroids;B: one or more biological agents selected from vaccines, cytokines,protein and peptide drugs or antibodies.
 18. The composition of claim17, wherein said alkylating agents are one or more selected fromcyclophosphamide, melphalan, temozolomide, carboplatin, cisplatin,and/or oxaliplatin.
 19. The composition of claim 17, wherein saidanti-metabolites are one or more selected from 5-fluorouracil,6-mercaptopurine, cytarabine, gemcitabine, and/or methotrexate.
 20. Thecomposition of claim 17, wherein said antitumor antibiotics are one ormore selected from actinomycin-D, bleomycin, daunorubicin, and/ordoxorubicin.
 21. The composition of claim 17, wherein said topoisomeraseinhibitors are one or more selected from etoposide, irinotecan,teniposide, and/or topotecan.
 22. The composition of claim 17, whereinsaid mitotic inhibitors are one or more selected from docetaxel,estramustine, paclitaxel, and/or vinblastine.
 23. The composition ofclaim 17, wherein said steroids are one or more selected fromprednisone, methylprednisolone, and/or dexamethasone.
 24. Thecomposition of claim 17, wherein said antibodies are selected fromHu3F8, hu14.18K322A, Hu14.18-IL-2, dinutuximab or any combinationthereof.
 25. The composition of claim 17, wherein said composition isformulated into one or more of the following administrative forms: aparenteral formulation, an aqueous solution, a liposome, an injectablesolution, an injectable suspension, an intravenous solution, anintravenous suspension/nanosuspension, a tablet, a pill, a lozenge, acapsule, a caplet, a patch, a spray, an inhalant, a powder, afreeze-dried powder, a patch, a gel, a geltab, a suspension, ananosuspension, a microparticle, a nanoparticle, a nanoliposome, amicrogel, a pellet, a suppository, an oral suspension, an oraldisintegrating tablet, an oral dispersible tablet, an oraldisintegrating film, a microemulsion, a nanoemulsion and aself-emulsifying drug delivery system and/or any combination thereof.26. A method for reducing the frequency or amount of a chemotherapeuticagent needed to treat a cancerous tumor and/or delay progression ofcancer from tumor-initiating cells, the method comprising: administeringa composition comprising an effective amount of a compound of Formula(I) or Formula (II):

or any pharmaceutically acceptable salt thereof, in combination with oneor more of chemotherapeutic agents, to a subject in need thereof, theone or more of the chemotherapeutic agents being selected fromalkylating agents, anti-metabolites, antitumor antibiotics,topoisomerase inhibitors, mitotic inhibitors, steroids and/or anymixtures thereof; wherein the frequency or amount of thechemotherapeutic agent administered is approximately up to 75 to 90percent less than a standard protocol without administration of thecomposition comprising an effective amount of the compound of Formula(I) or Formula (II).